Staphylococcus aureus is a major human pathogen that causes significant morbidity and mortality in both hospital- and community-acquired infections. The appearance of multidrug-resistant strains has compounded this problem, galvanizing efforts aimed at identifying novel therapeutic targets. One promising area for the development of novel antimicrobials is S. aureus heme metabolism, as heme acquisition is vital to staphylococcal pathogenesis. S. aureus acquires heme from the most abundant heme source in the host, hemoglobin contained within circulating erythrocytes. The process of heme uptake and metabolism is performed by the iron regulated surface determinant system (Isd). This is an application for continuation of a project to dissect the biochemical steps involved in S. aureus heme acquisition. Studies conducted during the present funding period indicate that S. aureus has evolved to preferentially recognize human hemoglobin over hemoglobin from other animal species. Once internalized, the intracellular fate of heme is dependent on the iron status of the bacterium. More specifically, iron starved S. aureus utilize heme as an iron source while heme is segregated intact to the bacterial membrane during iron replete conditions. Based on these fundamental discoveries, new studies are proposed to understand the mechanism and function of heme acquisition in S. aureus. Our current working model is that IsdB functions as the hemoglobin receptor that permits heme-iron uptake into the bacterial cytoplasm. During conditions of iron starvation, heme is degraded to free iron and an unprecedented small molecule which we have named staphylobilin. The regulation of heme degradation occurs through transcriptional and post-transcriptional mechanisms allowing S. aureus to tailor its heme utilization machinery to respond to changes in iron and heme levels. Under iron replete conditions, heme is not degraded but is instead used intact as a cofactor of membrane hemoproteins. This proposal focuses on testing this model for heme acquisition and metabolism in a series of three integrated Specific Aims. We will utilize genetics, inorganic chemistry, biochemistry, and animal infection experiments to (i) define the mechanism by which S. aureus preferentially utilizes human hemoglobin, (ii) determine the fate and function of heme and staphylobilin within bacterial cells, and (iii) provide a mechanistic understanding of how S. aureus regulates the Isd system in response to changes in nutrient availability. Results from these studies will yield a molecular blueprint of the heme-iron acquisition machinery in S. aureus, and permit the rational design of small molecule inhibitors for therapeutic intervention against S. aureus infection.